Reactivation of a Trypanosoma cruzi infection in a rhesus monkey (Macaca mulatta) experimentally infected with SIV.

Trypanosoma cruzi-like flagellates were incidentally noted in blood smears of a routinely monitored rhesus monkey experimentally infected with the simian immunodeficiency virus (SIV). Immunodeficiency in the course of the SIV infection reactivated a chronic infection of Chagas' disease that had been unnoticed when the macaque was imported to Europe. The animal developed no specific clinical symptoms of American trypanosomiasis, but histologically a chagasic myocarditis was detected. Analysis of the small subunit rRNA gene of the trypanosome identified the protozoan as T. cruzi.

Lack of simian immunodeficiency virus (SIV) specific IgA response in the intestine of SIV infected rhesus macaques.

BACKGROUND: Little is known about secretory immunity-the major defence mechanism at mucosal surfaces-in human immunodeficiency virus (HIV) infected patients, especially in the early stages of the disease. AIMS: The aim of the study was to analyse mucosal immunoglobulin production and simian immunodeficiency virus (SIV) specific antibody response in the intestinal mucosa during the course of SIV infection in comparison with serum and saliva. ANIMALS AND METHODS: IgG, IgA, and IgM concentrations were determined in supernatants of short term cultured duodenal biopsies, serum, and saliva from SIV infected rhesus macaques (n=8) and controls (n=2) by ELISA at defined times before and after infection. Specific antibodies to SIV were detected by western blot and/or dot blot analysis. In addition, rectal swabs from two uninfected and 12 SIV infected rhesus macaques (seven without and five with enteritis) were analysed for albumin and IgG concentrations. RESULTS: An increase in total intestinal IgG and a decrease in IgA were observed. SIV specific IgG or IgA responses were detectable as early as one week after SIV infection in the serum of seven of eight animals. In contrast, intestinal SIV specific IgG production was detected only four weeks after infection in six of eight animals, and intestinal SIV specific IgA was not produced in the intestine at any time point. In saliva, the secretory component on SIV specific IgA was only detected in one animal at week 24 after infection. Enteritis is frequent in SIV infected animals and results in a significant increase in albumin and IgG secretion into the intestinal lumen. CONCLUSION: Despite modest quantitative changes in mucosal immunglobulin production there was a total lack of SIV specific IgA synthesis in the intestine during SIV infection. This lack or disturbed secretory SIV specific IgA response at mucosal surfaces may explain the rapid and high HIV/SIV replication in this compartment. In addition, our investigations indicate secretion of serum proteins into intestinal fluids during SIV infection. Previous investigations using intestinal secretions or swabs for analysing quantitative and specific immunglobulins therefore should be interpreted with caution.

Efficient class I major histocompatibility complex down-regulation by simian immunodeficiency virus Nef is associated with a strong selective advantage in infected rhesus macaques.

Substitution of Y223F disrupts the ability of simian immunodeficiency virus (SIV) Nef to down-modulate major histocompatibility complex (MHC) class I from the cell surface but has no effect on other Nef functions, such as down-regulation of CD4, CD28, and CD3 cell surface expression or stimulation of viral replication and enhancement of virion infectivity. Inoculation of three rhesus macaques with the SIVmac239 Y223F-Nef variant revealed that this point mutation consistently reverts and that Nef activity in MHC class I down-modulation is fully restored within 4 weeks after infection. Our results demonstrate a strong selective pressure for a tyrosine at amino acid position 223 in SIV Nef, and they constitute evidence that Nef-mediated MHC class I down-regulation provides a selective advantage for viral replication in vivo.

Simian immunodeficiency virus in which nef and U3 sequences do not overlap replicates efficiently in vitro and in vivo in rhesus macaques.

The nef genes of human immunodeficiency virus and simian immunodeficiency virus (SIV) overlap about 80% of the U3 region of the 3' long terminal repeat (LTR) and contain several essential cis-acting elements (here referred to as the TPI region): a T-rich region, the polypurine tract, and attachment (att) sequences required for integration. We inactivated the TPI region in the nef reading frame of the pathogenic SIVmac239 clone (239wt) by 13 silent point mutations. To restore viral infectivity, intact cis-regulatory elements were inserted just downstream of the mutated nef gene. The resulting SIV genome contains U3 regions that are 384 bp shorter than the 517-bp 239wt U3 region. Overall, elimination of the duplicated Nef coding sequences truncates the proviral genome by 350 bp. Nonetheless, it contains all known coding sequences and cis-acting elements. The TPI mutant virus expressed functional Nef and replicated like 239wt in all cell culture assays and in vivo in rhesus macaques. Notably, these SIVmac constructs allow us to study Nef function in the context of replication-competent viruses without the restrictions of overlapping LTR sequences and important cis-acting elements. The genomes of all known primate lentiviruses contain a large overlap between nef and the U3 region. We demonstrate that this conserved genomic organization is not obligatory for efficient viral replication and pathogenicity.

Enhanced cellular immune response and reduced CD8(+) lymphocyte apoptosis in acutely SIV-infected Rhesus macaques after short-term antiretroviral treatment.

Losing the decisive virus-specific functions of both CD4(+) and CD8(+) T lymphocytes in the first weeks after immunodeficiency virus infection ultimately leads to AIDS. The SIV/rhesus monkey model for AIDS was used to demonstrate that a 4-week chemotherapeutic reduction of viral load during acute SIV infection of macaques allowed the development of a competent immune response able to control virus replication after discontinuation of treatment in two of five monkeys. Increasing SIV-specific CD4(+) T-helper-cell proliferation was found in all macaques several weeks after treatment, independent of their viral load. However, only macaques with low viral loads showed persistent T-cell reactivity of lymph node cells. In contrast to animals with higher viral loads, T-helper-cell counts and memory T-helper cells did not decline in the two macaques controlling viral replication. Lymphocyte apoptosis was consistently low in all treated macaques. In contrast, high CD8(+) lymphocyte death but only slightly increased CD4(+) lymphocyte apoptosis were observed during the first weeks after infection in untreated control animals, indicating that early apoptotic death of virus-specific CTL could be an important factor for disease development. Antiretroviral treatment early after infection obviously retained virus-specific and competent T lymphocytes, whereby a virus-specific immune response could develop in two animals able to control the viral replication after cessation of treatment.

Herpesvirus saimiri pathogenicity enhanced by thymidine kinase of herpes simplex virus.

Herpesvirus saimiri can be used as an efficient gene expression vector for human T lymphocytes and thus may allow applications in experimental leukemia therapy. We constructed recombinant viruses for the functional expression of the thymidine kinase (TK) of herpes simplex virus type 1 (HSV) as a suicide gene. These viruses reliably allowed the targeted elimination of transduced nonpermissive human T cells in vitro after the administration of ganciclovir. To test the reliability of this function under the most stringent permissive conditions, in this study we analyzed the influence of the prodrugs ganciclovir and acyclovir in common marmosets on the acute leukemogenesis induced by either wild-type herpesvirus saimiri C488 or by a recombinant derivative expressing TK of HSV. Antiviral drug treatment did not influence the rapid development of acute disease. In contrast, the presence of the HSV tk gene resulted in a faster disease progression. In addition, HSV TK-expressing viruses showed faster replication than wild-type virus in culture at low serum concentrations. Thus, HSV TK accelerates the replication of herpesvirus saimiri and enhances its pathogenicity. This should be generally considered when HSV TK is applied as a transgene in replication-competent DNA virus vectors for gene therapy.

Total numbers of lymphocyte subsets in different lymph node regions of uninfected and SIV-infected macaques.

Human immunodeficiency virus (HIV) infection leads to a decline of CD4+ T-cells in blood. Because blood represents only a small proportion of the total lymphocyte pool, it is important to investigate other lymphoid organs. So far, only relative proportions of lymphocyte subsets in single peripheral lymph node (LN) regions of HIV-infected patients and simian immunodeficiency virus (SIV)-infected macaques have been documented. We have therefore quantified the absolute numbers of lymphocyte subsets in blood and six different LN regions of 10 uninfected and 26 SIV-infected macaques. In addition, we have determined the expression of markers of activation and differentiation. Already, in uninfected monkeys, there were significant differences in the cellular composition of different LN regions. Infection with SIV resulted in drastic changes in the proportion as well as absolute numbers of different lymphocyte subsets. Moreover, the relative contribution of the single LN regions to the total lymphocyte pool was also altered.

Packaging of small molecules into VP1-virus-like particles of the human polyomavirus JC virus.

Recombinantly expressed VP1-virus-like particles (VP1-VLP) of human polyomavirus JC virus (JCV) were described recently as a new DNA transporter system. It was shown that DNA molecules could be packaged into VP1-VLP during a controlled chemical reassociation/dissociation process. In the present study VP1-VLP were studied as carriers for pharmaceutical substances. Propidium iodide (PI) was packaged into VP1-VLP as a reporter molecule. The PI-containing VP1-VLP could be detected directly by flow cytometry. The fluorescence intensity of the VP1-VLP depended strongly on the initial PI concentration. This packaging method is easy to handle and applicable to viruses and VP1-VLP which can be dissociated and reassociated chemically.

Homozygosity for a conserved Mhc class II DQ-DRB haplotype is associated with rapid disease progression in simian immunodeficiency virus-infected macaques: results from a prospective study.

In human immunodeficiency virus type 1 (HIV-1)-infected individuals, disease progression varies considerably. This is also observed after experimental infection of macaques with simian immunodeficiency virus (SIV). Major histocompatibility complex (MHC) genes may influence disease progression in both species. Homozygosity for Mhc-Mamu (Macaca mulatta)-DQB1*0601 was previously identified to be associated with rapid disease progression in SIV-infected macaques. To validate the association of this genotype with disease progression, a prospective study was carried out. Six unrelated monkeys homozygous for Mamu-DQB1*0601 and DRB1*0309-DRB*W201 and 6 heterozygous monkeys were infected with SIVmac. Five of the homozygous and only 1 of the heterozygous monkeys died rapidly after infection, with manifestations of AIDS. These results were validated by a retrospective survival analysis of 71 SIV-infected monkeys. The identified DQ-DRB genotype is frequent among monkeys of different breeding colonies and allows a fairly reliable selection before infection of monkeys predisposed for rapid disease progression.

Simian immunodeficiency virus containing mutations in N-terminal tyrosine residues and in the PxxP motif in Nef replicates efficiently in rhesus macaques.

SIVmac Nef contains two N-terminal tyrosines that were proposed to be part of an SH2-ligand domain and/or a tyrosine-based endocytosis signal and a putative SH3-ligand domain (P(104)xxP(107)). In the present study, we investigated the effects of combined mutations in these tyrosine and proline residues on simian immunodeficiency virus (SIV) Nef interactions with the cellular signal transduction and endocytic machinery. We found that mutation of Y(28)F, Y(39)F, P(104)A, and P(107)A (FFAA-Nef) had little effect on Nef functions such as the association with the cellular tyrosine kinase Src, downregulation of cell surface expression of CD4 and class I major histocompatibility complex, and enhancement of virion infectivity. However, mutations in the PxxP sequence reduced the ability of Nef to stimulate viral replication in primary lymphocytes. Three macaques infected with the SIVmac239 FFAA-Nef variant showed high viral loads during the acute phase of infection. Reversions in the mutated prolines were observed between 12 and 20 weeks postinfection. Importantly, reversion of A(107)-->P, which restored the ability of Nef to coprecipitate a 62-kDa phosphoprotein in in vitro kinase assays, did not precede the development of a high viral load. The Y(28)/Y(39)-->F(28)/F(39) substitutions did not revert. In conclusion, mutations in both the tyrosine residues and the putative SH3 ligand domain apparently do not disrupt major aspects of SIV Nef function in vivo.

Co-receptor usage of BOB/GPR15 in addition to CCR5 has no significant effect on replication of simian immunodeficiency virus in vivo.

Human immunodeficiency virus type 2 (HIV-2) and the closely related simian immunodeficiency viruses (SIVs) frequently use the orphan receptor BOB/GPR15 in addition to the chemokine receptor CCR5 for efficient entry and replication. However, the role of BOB/GPR15 in replication and pathogenesis of HIV-2 and SIV in vivo is unclear. This study shows that a single amino acid substitution in the V3 loop of the pathogenic SIVmac239 clone, 321P-->S, impaired the ability to use BOB/GPR15 for entry and replication but had little effect on the ability to use CCR5. This envelope variant replicated with an efficiency comparable with the parental SIVmac239 isolate in rhesus macaques. Furthermore, the mutant genotype and phenotype remained stable even after the onset of immunodeficiency. These results suggest that this cofactor plays only a minor role for the pathogenicity of the HIV-2/SIVmac/SIVsm group of primate lentiviruses.

The human immunodeficiency virus type 1 nef gene can to a large extent replace simian immunodeficiency virus nef in vivo.

The nef gene of the pathogenic simian immunodeficiency virus (SIV) 239 clone was replaced with primary human immunodeficiency virus type 1 (HIV-1) nef alleles to investigate whether HIV-1 Nef can substitute for SIV Nef in vivo. Initially, two rhesus macaques were infected with the chimeric viruses (Nef-SHIVs). Most of the nef alleles obtained from both animals predicted intact open reading frames. Furthermore, forms containing upstream nucleotide substitutions that enhanced expression of the inserted gene became predominant. One animal maintained high viral loads and slowly progressed to immunodeficiency. nef long terminal repeat sequences amplified from this animal were used to generate a second generation of Nef-SHIVs. Two macaques, which were subsequently infected with a mixture of cloned chimeric viruses, showed high viral loads and progressed to fatal immunodeficiency. Five macaques received a single molecular clone, named SHIV-40K6. The SHIV-40K6 nef allele was active in CD4 and class I major histocompatibility complex downregulation and enhanced viral infectivity and replication. Notably, all of the macaques inoculated with SHIV-40K6 showed high levels of viral replication early in infection. During later stages, however, the course of infection was variable. Three animals maintained high viral loads and developed immunodeficiency. Of the remaining two macaques, which showed decreasing viral loads after the acute phase of infection, only one efficiently controlled viral replication and remained asymptomatic during 1.5 years of follow-up. The other animal showed an increasing viral load and developed signs of progressive infection during later stages. Our data demonstrate that HIV-1 nef can, to a large extent, functionally replace SIVmac nef in vivo.

Rapid infection of oral mucosal-associated lymphoid tissue with simian immunodeficiency virus.

The early events during infection with an immunodeficiency virus were followed by application of pathogenic simian immunodeficiency virus atraumatically to the tonsils of macaques. Analyses by virologic assays and in situ hybridization revealed that the infection started locally in the tonsils, a mucosal-associated lymphoid organ, and quickly spread to other lymphoid tissues. At day 3, there were few infected cells, but then the number increased rapidly, reaching a high plateau between days 4 and 7. The infection was not detected in the dendritic cell-rich squamous epithelium to which the virus was applied; instead, it was primarily in CD4+ tonsillar T cells, close to the specialized antigen-transporting epithelium of the tonsillar crypts. Transport of the virus and immune-activating stimuli across this epithelium would allow mucosal lymphoid tissue to function in the atraumatic transmission of immunodeficiency viruses.

Pulmonary ischemia/reperfusion injury: a quantitative study of structure and function in isolated heart-lungs of the rat.

Early graft dysfunction after lung transplantation is a significant and unpredictable problem. Our study aimed at a detailed investigation of structure-function correlations in a rat isolated heart-lung model ofischemia/ reperfusion injury. Variable degrees of injury were induced by preservation with potassium-modified Euro-Collins solutions, 2 hr of cold ischemia, and 40 min of reperfusion. Pulmonary artery pressure (Ppa), pulmonary vascular resistance (PVR), peak inspiratory pressure (PIP), and perfusate gases (deltaPO2, deltaPCO2) were recorded during reperfusion. Right lungs were used to calculate W/D-weight ratios. Nineteen experimental and six control left lungs were fixed for light and electron microscopy by vascular perfusion. Systematic random samples were analyzed by stereology to determine absolute and relative volumes of lung structures, the amount of interstitial and intraalveolar edema, and the extent of epithelial injury. Lectin- and immunohistochemistry using established epithelial cell markers were performed in three animals per group to reveal sites of severe focal damage. Experimental lungs showed a wide range in severity of ischemia/ reperfusion injury. Intraalveolar edema fluid amounted to 77-909 mm3 with a mean of 448+/-250 mm3 as compared with 22+/-22 mm3 in control lungs (P<0.001). Perfusate oxygenation (deltaPO2) decreased from 30.5+/-15.2 to 21.7+/-15.2 mm Hg (P=0.05) recorded after 5 and 40 minutes of reperfusion. In experimental lungs, a surface fraction of 1% to 58% of total type I pneumocyte surface was damaged. Intraalveolar edema per gas exchange region (Vv ape,P) and deltaPO2 were related according to deltaPO2 = 96 - 60 x log10(Vv ape,P) [mm Hg]. The extent of epithelial injury did not correlate with deltaPO2 nor with intraalveolar edema, but increased significantly with PVR. Lectin- and immunohistochemistry revealed focal severe damage to the alveolar epithelium at the border of perivascular cuffs.

Rapid mucosal CD4(+) T-cell depletion and enteropathy in simian immunodeficiency virus-infected rhesus macaques.

BACKGROUND & AIMS: Human immunodeficiency virus (HIV) infection leads to severe immunologic and functional disturbances in the intestinal tract in late stages of the disease. Information on mucosal pathology directly after infection is limited. We characterized this early phase in rhesus macaques infected with simian immunodeficiency virus (SIV). METHODS: Eight rhesus macaques were infected with SIV. Upper endoscopy was performed at defined times before and after infection. Viral load, percentage of CD4(+) T cells, villus height, crypt depth, and Ki-67-positive crypt cells were analyzed in duodenal biopsy specimens. Serum beta-carotene and vitamin D levels were assessed. RESULTS: A rapid increase of duodenal SIV core protein (p27) concentration and an almost complete loss of intestinal CD4(+) T cells was found within 2 weeks after infection. A decrease of villus height was observed, and the percentage of Ki-67-positive (proliferating) crypt cells increased. Serum concentrations of vitamin D decreased in 6 of 8 animals, and beta-carotene concentrations decreased in 3 of 8 animals after infection. CONCLUSIONS: Mucosal SIV replication and intestinal CD4(+) T cell depletion are early events in SIV-infected rhesus macaques. The structural changes of the mucosa strongly support the concept of HIV/SIV-induced enteropathy. In contrast to late-stage human HIV infection, early small intestinal villous atrophy in SIV infection is associated with crypt hyperplasia.

Intra-nasal infection of macaques with Yellow Fever (YF) vaccine strain 17D: a novel and economical approach for YF vaccination in man.

Investigating new and simple application routes for YF vaccine, four groups of 4-6 rhesus monkeys were vaccinated with live attenuated 17D YF-vaccine. In two groups the vaccine was administered either as spray into the oral cavity, or as an encapsulated form directly into the stomach. Only one out of eight animals developed a humoral immune response against 17D. In the third group receiving the vaccine intranasally by spray and in the fourth group serving as control all ten monkeys developed an immune response. From all except one of these seroconverted monkeys virus could be detected either by virus reisolation or RT-PCR. All these animals showed a serological immune response in immunofluorescence and neutralisation test. Parallel to viremia, an increase of neopterin as an unspecified immune activation marker could be demonstrated for these animals. Intra-nasal application of 17D-vaccine seems to be a good alternative to subcutaneous immunisation in mass vaccination campaigns.

Immunohistochemical studies of productive rhesus cytomegalovirus infection in rhesus monkeys (Macaca mulatta) infected with simian immunodeficiency virus.

In humans infected with the human immunodeficiency virus (HIV), clinical disease due to human cytomegalovirus (HCMV) infection is one of the AIDS-defining diseases; HCMV is the most common opportunistic infection found postmortem. Histologically, the typical lesions are characterized by "owl's eye cells." In rhesus monkeys infected with simian immunodeficiency virus (SIV), comparable lesions are caused by an infection with the rhesus CMV (RhCMV). The aim of this study was to investigate the incidence of productive and latent RhCMV infection in monkeys infected with SIV macaques (SIVmac). Eleven SIVmac-infected rhesus monkeys, which were euthanatized after developing AIDS-like disease, and 11 clinically healthy and uninfected animals comprised the study. The monkeys were screened serologically for RhCMV by western-blot analysis. Immunohistochemistry was performed by an indirect immunoperoxidase technique with a polyclonal rabbit RhCMV-antiserum. Lesions characteristic of RhCMV-associated diseases were detected histologically. All animals were latently RhCMV-infected. Seven of eleven (63.6%) SIV-infected macaques were productively RhCMV infected according to immunohistochemistry. RhCMV antigen was identified in the gastrointestinal tract, the hepatobiliary system, the lungs, and the testicles. Two of these seven animals showed characteristic inflammatory lesions associated with productive infection. Consequently, the CMV prevalence in SIVmac-infected rhesus monkeys and human AIDS patients is comparable.

Effects of ischaemia and preservation on the ultrastructure of the bronchiolar epithelium. A quantitative electron microscopic study of human and canine lungs.

In ten cases of clinical human single-lung transplantation, the nontransplanted Euro-Collins-preserved contralateral lungs were examined using electron microscopy to determine the effects of ischaemia on the bronchiolar epithelium. Existing structural damage at the time of transplantation was characterized using this approach, and nine nonpreserved canine single lungs were also investigated to identify the impact of ischaemia. The study revealed a significant correlation between the duration of ischaemia and the mitochondrial surface-to-volume ratio, which can serve as a morphometric criterion for mitochondrial damage, in canine lungs. However, this correlation was not found in the human donor lungs. Further examination of human donor lungs showed slight to moderate damage to the endoplasmic reticulum and nuclear chromatin. In addition, various degrees of damage to mitochondrial structure, ranging from inconspicuous to severe, were found. The mitochondrial surface-to-volume ratio can be considered to be a suitable criterion for the quantification of ischaemic damage of the bronchiolar epithelium under experimental conditions. Ultrastructural analysis of human donor lungs revealed intact bronchiolar epithelial cell structures at the time of transplantation, reflecting adequate organ preservation with Euro-Collins solution.